ARAF - The Ultimate Comfort!

Samples were then fixed with 1% OsO4 in phosphate buffer for 30 min and rinsed briefly with distilled water, followed by staining with 1% uranyl acetate and dehydrated in a series of ethanol solutions (50, 70, 90, and 100%) for 5 min each. After two more additional changes in 100% ethanol, the samples were embedded in epoxy resin and polymerized at 60��C for 24 h. Polymerized resin blocks were sectioned into ?60 nm slices and post-stained with uranyl acetate and lead citrate. Samples were visualized using a Philips/FEI (Morgagni) transmission electron microscope with a Gatan digital camera. GeoChip Analysis The Powersoil? DNA Isolation Kit from MO BIO Laboratories, Inc. (Carlsbad, CA, USA) was used to extract genomic DNA from each sample. DNA extracted from the triplicates under each condition (with/without Ag-NPs) were pooled, respectively. Pooled DNA (1 ��g)was labeled with Cy3 and hybridized to the GeoChip 4 microarray synthesized by NimbleGen (Madison, WI, USA) and processed as previously described by Lu et al. (2012). The signal-to-noise ratio threshold for a spot to be considered positive was ��2 as described previously (He et al., 2010). Pearson��s correlation coefficient (r) was calculated as a measure of the similarity between selected gene profiles (Pearson, 1896). That is, for two profiles of normalized gene signal intensity: X = xi : i = 1,��, n for no treatment control and Y = yi : i = 1,��, n for Ag-NP treated sample, r=��i=1n(xi?x?)(yi?y?)��i=1n(xi?x?)2.��i=1n(y?y?)2, (1) where x?=1/n??��i=1n??xi and y?=1/n??��i=1n??yi qPCR Analysis qPCR was used to quantify total bacteria and bacteria associated with nitrification and denitrification. A CFX 96 real-time PCR system with a C1000 Thermal cycler (Bio-Rad Laboratories, Inc.) was used to run the reactions. 10 ��L of SsoFast EvaGreen Supermix (Bio-Rad Laboratories, Inc.), 10 pmol of each primer, 6 ��L of sterile water, and 2 ��L of DNA template (7 ��L of sterile water, and 1 ��L of DNA template for total bacteria) were added to each 20 ��L reaction system. Primers used and reaction programs are shown in Table ?Table11 Calibration was performed with serial dilutions of a known quantity of the target fragments. Triplicate reactions were run for all samples analyzed. Melting curves were examined to eliminate primer dimer ARAF formation or non-specific amplification. Table 1 qPCR primers and conditions. Results Uptake of Ag-NPs into the Biofilm and Cells Ag-NPs were incorporated into the biofilms quickly after the incubation started. In the abiotic sample in Figure ?Figure1A1A, most Ag-NPs are round with a diameter no more than 20 nm and some formed aggregates larger than 50 nm. No particles similar to the Ag-NPs were seen in the control biofilm (Figure ?Figure1B1B). After 45 min, Ag-NPs (Figures 1C,D, white arrows) were observed in the biofilm and only smaller Ag-NPs entered the biofilms.